Alzheimer's disease (AD) is one of the most significant diseases of aging. It is characterized clinically by the progressive loss of short- term memory and decline in other cognitive functions. Although its etiology has not been established, a number of plausible hypotheses have been proposed. Three of the proposed etiologies that are directly relevant to this proposal are (1) abnormal processing of amyloid precursor protein to yield reactive, neurotoxic fragments, (2) selective failure in energy metabolism, and (3) enhanced oxidative stress, leading to Ca2+ ion loading. These toxic mechanisms will be studied in both tissue samples obtained from rat and human brains and in primary cultures of astrocytes and neurons obtained from E-18 rat brain regions and from cultured astrocytes from brain regions obtained at autopsy. Tissue membrane and protein status using synaptosomes from rat hippocampus, neocortex and cerebellum and a series of brain regions (both affected and unaffected in AD) obtained from both AD and age matched control brain tissue obtained at autopsy will be characterized for changes induced by amyloid peptides and the other reactive oxygen species (ROS) generators. The effect of prior, non-lethal oxidative stress on susceptibility to neurotoxic ROS levels will be determined for rat and human brains regions and for tissue culture samples. We will use EPR spectroscopy and assays for enzyme activity and protein carbonyl. Preparations will be exposed in vitro to the same ROS generators: amyloid peptides that have been characterized for radical production, Fe2+/hydrogen peroxide and xanthine/xanthine oxidase. In vitro studies will be conducted to determine the effects of ROS generators on viability, Ca2+ loading, enzyme status and protein carbonyl staining using confocal microscopy quantitation. Selective failure in energy metabolism will be studied in vivo using aged Fisher-344 (F-344), hyperoxic exposed F-344 rats, ROS peroxide exposed tissue cultures to produce oxidative stress and a reduced creatine kinase (CK) activity level that is comparable to normal aged human brain. In vitro approaches using antisense oligonucleotides to reduce anti-oxidant and metabolic status (Cu-Zn SOD, Mn-SOD and the different isoforms of CK) in separate cultures of E-18 cortex, hippocampus and cerebellum containing either astrocytes, neurons or mixed neuronal/astrocyte cultures will be used. In addition, we will attempt to culture astrocytes from selected brain regions of neurologically normal and AD brains. We will determine the level of CK, SOD, catalase and glutathione peroxidase mRNA and protein in cultured astrocytes using in situ hybridization, Northern, Western Analyses and enzyme activity assays. The hypothesis that CK is a metabolic buffer to prevent ROS production via xanthine oxidase will be tested.